Cell-free reduction of carboxylic acids with secreted carboxylic acid reductase.

Mycobacterium marinum CAR (Mm CAR) is one of the most widely used CARs as the key enzyme for the synthesis of aldehydes, alcohols and further products from the respective carboxylic acids. Herein, we describe the first functionally secreted 131 kDa CAR and its isolated A-domain using Komagataella phaffii and a methanol-free constitutive expression strategy. Precipitated and lyophilized Mm CAR (500 µg) was isolated from the culture supernatant and showed no decrease in activity for piperonylic acid (80% conversion), even when stored for up to 3 weeks at 4°C. Lyophilized Mm CAR precipitate gave 48% yield of E/Z -nonanal-4-nitrobenzoyloxime from the reduction of nonanoic acid and in-situ derivatization with O -4-nitrobenzoyl-hydroxylamine. Furthermore, K. phaffii could successfully secrete the Mm CAR adenylation domain. Its activity was confirmed by the amidation of benzoic acid with n -hexylamine. Neither enzyme variant was glycosylated by the yeast. In summary, functional CAR can be secreted by K. phaffii and used for cell free conversion of carboxylic acids to various products. [Display omitted] • Active full-length Mm CAR and Mm CAR A-domain were secreted by Komagataella phaffii for the first time. • Mm CAR A-domain was active for amidation of benzoic acid with n-hexylamine. • Precipitated, lyophilized full-length Mm CAR can be stored without activity loss at 4°C for at least 3 weeks. [ABSTRACT FROM AUTHOR]